Compositions for improvement of wound healing

ABSTRACT

This invention relates to aqueous solutions of reactive chlorine compounds having the empirical formulae H 2 Cl 2 O 6  or ClO 3 H, for example, and the derivatives, anions or salts thereof. The invention further relates to methods for the production of said compounds and the use thereof in the pharmaceutical and particularly in the medical field, in cosmetics, medicinal care and in the domains of food technology and technology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. Ser. No. 12/574,849 filed Oct. 7, 2009,which is a continuation of U.S. Ser. No. 10/580,392, which is the U.S.national phase of PCT/EP2004/013212 filed Nov. 22, 2004, which in turnclaims the priority of DE 103 54 768.1 filed Nov. 21, 2003, the entirerespective disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to reactive chlorine compounds such as dichloricacids, the intermediate product peroxochloric acid as well asperoxochlorous acid and their individual derivatives, anions, and/orsalts. It further relates to processes for manufacturing these compoundsand their use in the pharmaceutical field, here in particular, inmedical treatment as drugs and disinfectants, in the fields of cosmeticsand medicinal care as histocompatible deodorants, in the field offoodstuff treatment and technology, in particular in the preservation offoods and beverages, as a bleaching agent and for drinking waterdisinfection, in the antimicrobial treatment of plants and fruits inagriculture, and as an oxidizing agent in technical chemistry and forcleaning waste gas.

2. Related Technology

Oxidizing agents have a very wide range of applications in technicalchemistry, in hygiene and in food preservation, in cosmetics and also inpharmaceutical uses.

According to Polly Matzinger (Polly Matzinger: “Tolerance, Danger, andthe Extended Family” in Annu. Rev. Immunol. 1994, 12) cells dying due toviolence, i.e. through massive radiation effects, toxic substances,parasitic, bacterial or viral infective agents, lytic, non-apoptoticeffects, emit danger signals. These must persist so that the body's owndefences, which as well as the actual antigen signal require anon-specific co-stimulation from antigen-presenting cells (e.g.macrophages), can have an optimum clinical effect.

During a violent, non-apoptotic cell death, phagocytes (so-called microand macrophages) are responsible for cellular debris disposal. In thisdebris disposal process oxidatively effective oxygen metabolites arereleased. Hydrogen peroxide (H₂O₂) is the most well-known of thesesubstances. In-vitro-trials show that, in the micromolar range, H₂O₂ canlead to an immune modulation of lymphocytes via the activation of thetranscription factor HF-kappa B (R. Schreck et al., The EMBO Journal10(8), 2247-58 (1991); M. Los et al., Eur. J. Immunol. 25, 159-65(1995). The working group of Avraham Novogrodsky was the first todemonstrate in vitro that certain oxidizing agents (Bowers W. E.:“Stimulation of Lymphocytes with Periodate or Neuraminidase plusGalactose Oxidase—NAGO” p. 105-109, Review in Immunochemical TechniquesPart K Methods in Enzymology Vol. 150, 1987), among other effects, alsoincrease the H₂O₂ formed in the body itself co-mitogenically bylymphocyte proliferation due to antigen stimulation, if macrophages aresimultaneously present in the lymphocyte culture (Stenzel K. H., RubinA. L., Novogrodsky A.: “Mitogenic and Comitogenic Properties of Hemin.”J. Immunol. 127, 6: 2469-2473 et ibid. cit. ref.). An immune responsewill be incomplete or not even take place at all if the oxidativelyeffective oxygen metabolites are not formed in sufficient quantities inthe body. Thus a tolerance or pathological anergy results. If themetabolites are produced excessively or for a disproportionately longperiod, then chronic inflammation and tissue scars will form.

As a result of these findings, one can assume that oxidatively effectiveoxygen compounds will have a therapeutic effect, particularly in suchclinical situations where their endogenous formation is insufficient ordeteriorates before the body injuries have completely healed and theinfective agents have been totally removed. A treatment success isexpected especially in those cases where the cells are indeed affectedby the infection but not destroyed and therefore do not emit “dangersignals”. Exemplary here are infections with leprosy and tuberculosisbacilli as well as infections caused by herpes and AIDS (HIV) viruses.

A report was published as early as 1906 on the successful clinical useof potassium bichromate in the healing of ichorous chronic wounds(Fenwick, J.: “The Treatment of Cancer by the Use of PotassiumBichromate”, British Medical Journal, Mar. 6, 1909, 589-591).

Further numerous publications, which have appeared in the meantime, showthat hydrogen peroxide formed physiologically within the body—as well asthe in vivo even more short-lived peroxonitrite which can also form fromthe equally physiological nitroxide and hydrogen peroxide—alsodemonstrates wound healing effects, whereby a positive immunomodulationplays an essential role. For example, the EPA-0390829 describes a methodfor increasing the syngenic intradermal cell proliferation through humangrowth factors using hydrogen peroxide injections. Such a comitogenicincrease in the growth factor effects of interleukin-2 was alsodescribed for periodate in 1987 (Wang J. et al., The American Journal ofMedicine 1987, 83: 1016-1023).

It is known that (co)mitogenic oxidants have intolerable side effects,such as e.g. for bichromate:—the now recognized carcinogenic effect ofchromium oxide: For periodate:—iodine hypersensitivity and toxiceffects. Therefore, their clinical use has to take place laboriously asan “adoptive transfer”, i.e. the blood cells are taken out, treated invitro and then returned in vivo—as described in the previously quotedstudy by J. Wang et al. 1987. For NAGO side effects are:—the foreignprotein sensitization: For H₂O₂:—the formation of toxic oxygen radicals.Here too, there are also technical problems concerning their use asdrugs, e.g. for H₂O₂:—short storage life in diluted aqueous solution;the catalase lability with massive oxygen gas release. For oxidizedubichinon derivatives problems are:—pharmaceutical manufacturingproblems and limited bioavailability.

Therefore, it was not possible up to now to transfer the experimentallydemonstrated immunopharmacological action of (co)mitogenic oxidants inclinical practice into tissue regeneration/wound healing, infectionresistance and the strengthening of the immune response. In clinicalpractice, as well as a local application, a systemic treatment, usuallyin the form of an intravenous administration, is also desirable.

Theo Gilbert Hinze (US 2003/0133878 A1, “American Composition for thetreatment of legionella pneumophila and a method for such treatment”)processed aqueous solutions of NaCl or KCl₂ (presumably the latterchemical formula here is a printing error) with electrochemicaloxidation at pH 6.5-7.5. It was conjectured that, as well as other ions,only the Cl₂O₆ ²⁻ ion could be present which at that time had beendescribed only in the preceding invention. This dimer contains thechlorine atoms in the +3 and +5 valence states.

The patent literature contains descriptions of a few furtherchlorine-oxygen preparations which are particularly used in suchtechnical fields where they serve as oxidants not only in industrialtechnology as bleaching agents and deodorants, but also where they arerecommended for paramedical applications such as in cosmetics for skinand hair care, for household cleaning, in the sanitary sector forhygiene and/or as disinfectants for surfaces (U.S. Pat. Nos. 2,701,781;3,123,521) and/or wounds (U.S. Pat. No. 4,084,747; EP-A-0 744 895), aspreservation agents for cheese (U.S. Pat. No. 3,147,124) and for theconditioning of drinking and bathing water (U.S. Pat. No. 4,296,103;DE-A-44 05 800, DE-A-19 518 464; WO 96/33947; WO 97/06098). The U.S.Pat. No. 4,296,103, EP-A-0 136 309, U.S. Pat. No. 4,507,285 andEP-A-0255145 describe the medical application of chlorine-oxygenpreparations.

WO 00/48940 contains a description of the preparation of achlorohydroperoxide with the formula HOOClO₂ where the chlorine hasvalence of 5. This hydroperoxide behaves as an acid which supplies theanion O₂ClOO⁻ in an aqueous environment. Therefore, it was calledperoxochloric acid and its anion is called peroxochlorate. It isreported that the combination of two peroxochlorate ions, underseparation of an oxygen molecule, can lead to derivatives ofperoxochlorate with one peroxo group and two chlorine atoms withdifferent valences. This ion is allocated the empirical formula(Cl₂O₆)²⁻.

It is disclosed that it would be possible to manufacture stableperoxochloric acid and stable salts or anions thereof in solution. Forexample, these compounds are obtained in aqueous solution by thereaction of chlorine dioxide with hydrogen peroxide if the work iscarried out at pH values which are equal to, or greater than, the pKsvalue of peroxochloric acid (HOOClO₂). pH values of 6.5 and more arepreferable, and the pH range of 10-12 is especially preferable.

Thus, in WO 00/48940 peroxochloric acid or its salts, peroxochloric acidand its salts or anions in aqueous solution, oligomeric derivatives ofthe peroxochlorate with mixed-valent chlorine atoms and their salts oranions in aqueous solution as well as the carbon dioxide adduct as anacid, an anion in solution or as a salt are disclosed.

In the meantime, it has been proved that an isolation of a crystallinemetallic salt of peroxochlorate, according to the specifications givenin WO 00/48940, is unsuccessful.

Due to the low concentrations of peroxochlorate in the preparationsmanufactured according to the specifications of WO 00/48940, it is onlypossible to prepare the deoxo dimers to a limited degree.

Svensson and Nelander published the preparation of HOOClO₂ at lowtemperatures of 17K (−256, 15° C.) in J. Phys. Chem. A 1999, 103,4432-4437.

Therefore, all the published chlorine-oxygen preparations do not fulfilthe requirement criteria of modern drug approval. These state that thepharmacodynamics of the preparation must be allocatable to a chemicallydefined compound as the so-called active substance which is to bestandardized as the pharmaceutical product. This is also necessary inorder to guarantee homogeneous drug quality.

The intrinsically good chlorine-oxygen compounds of WO 00/48940, and inparticular the deoxo dimer defined there, can, up to now, only bemanufactured to a limited degree. Therefore, a commercial exploitationappears to be impossible.

GENERAL DESCRIPTION OF THE INVENTION

The invention provides an oxidant without the disadvantages describedabove. As well as the usual technical, medicinal and disinfectant fieldsof application, such an oxidant should also offer the possibility offormulation as a medicament for both local and systemic treatment, e.g.for intravenous application as, for example, a drug for tissueregeneration, for wound healing and against infections or for enhancingthe immune response. Furthermore, it should fulfill the requirements ofmodern new drug approval procedures.

Particularly, therefore, the invention provides a further improvedoxidant and an improved process for its manufacture and application.

Surprisingly, it has become evident that this object can be solvedthrough the preparation of reactive chlorine compounds such as dichloricacids, the intermediate product peroxochloric acid as well asperoxochlorous acid, as well as their individual derivatives, anionsand/or salts.

The novel dichloric acids, according to the invention, are shown in thefollowing Table 1. Among these dichloric acids, the acids numbered No 1to No. 3 are particularly preferred embodiments of this invention.

TABLE 1 Formal oxidation numbers of No. chlorine Structural formula ofthe acid Structural formula of the dianion 1 +5, +5

2 +6, +4

3 +5, +5

4 +5, +3

As well as the valence pairs already described previously +3/+5 (WO00/48940) and +4/+4 (Bogdanchikov et al.), the dichloric acids accordingto the invention No. 1 to No. 3 with valences of +6/+4 and +5/+5 forchlorine were manufactured for the first time according to the processof the invention. The anion of the acid of No. 4 is described in WO00/48940. The manufacturing process described there, however, does notwork.

In WO 00/48940 the postulation was made that the deoxo dimer is formedfrom two molecules of a reactive chlorine-oxygen species(peroxochlorate) via the reaction2^(−OOClO) ₂→Cl₂O₆ ²⁻+O₂,

whereby the chlorine atoms are present in the oxidation numbers +3 and+5. However, the manufacture of a stable compound according to example 6of WO 00/48940, which is desirable under pharmaceutical law aspects, isnot successful.

The formation of the dimeric derivative from 2 molecules ofperoxochlorate according to the formula2^(−OOClO) ₂→Cl₂O₆ ²⁻+O₂

can namely only be expected at very high concentrations ofperoxochlorate (roughly from 2 to 3 mol/L). Such high concentrations,however, are impossible in practice due to the high instability of thecompound.

However, the examinations which led to the invention show that thereaction of peroxochlorate ions O₂ClOO⁻ with chlorite ions (ClO₂ ⁻)leads surprisingly directly to the palette of “dimeric” Cl₂O₆ ²⁻species:O₂ClOO⁻+ClO₂ ⁻→Cl₂O₆ ²⁻->->and isomers

Furthermore, surprisingly, with the help of the process according to theinvention, the preparation of the previously unknown peroxochlorite ion,O═ClOO⁻ and the peroxochlorous acid O═ClOOH derived from it issuccessful—in particular in the solutions containing chlorite accordingto the invention.

These chlorine compounds have not been described previously.

Especially round about the point of neutrality, the dissociation of thedichlorine species Cl₂O₆ ²⁻ into chlorate ions ClO₃ and peroxochloriteions OClOO⁻ is a clearly competitative reaction to the describedintramolecular redox reactions of the dichlorine species which lead tothe compounds 1-4 in the above table.

Insofar as reference is made to anions in the disclosure, the presenceof the necessary counterions (particularly in solution) is included aswell. The term “anions” is used in particular to stress that, insolution, the dichlorate is the more stable form compared with theprotonated acid. However, the term “anion” can, according to theinvention, and depending on the context, also be used in place of acid.The term “acid” can equally be used in place of “anion”.

The invention also relates to the process of manufacturing preparationswhich contain the dichloric acids and their derivatives, anions and/orsalts, and/or the peroxochlorous acid according to the invention and itsderivatives, anions and/or salts.

If one carries out the following steps, one can, in an amazingly simpleway, manufacture the dichloric acids and the peroxochlorite ionaccording to the present invention.

Chlorine dioxide reacts with an aqueous solution or water-containingsolution of hydrogen peroxide or another hydroperoxide or peroxide at apH value of ≧6.5,

the pH value is lowered by adding an acid,

the gaseous free reactive chlorine compound, preferably the protonatedperoxochlorine compound, is expelled with a cooled gas and collected inan alkaline solution,

the collected free reactive chlorine compound, preferably theperoxochlorine compound, is incubated at a pH between 6 and 8,preferably about 7 with an up to 100-fold excess, preferably up to a10-fold chlorite excess.

The dichloric and peroxochlorous acids of the invention, and also theions which are present at physiological pH values can therefore,according to the invention, also be present as a mixture withperoxochlorate and chlorite in solution. Such a solution containingdichloric acids, peroxochlorous acid, peroxochlorate and chloriteaccording to the invention, therefore counts among the particularlypreferable experimental practice examples of the invention.

In WO 00/48940, in contrast, chlorite-free solutions were produced inwhich the dichloric acids and the peroxochlorous acid of the inventionare not contained, or, chlorite-containing preparations were producedwhich contained practically only chlorite so that they are unsuitablefor pharmaceutical applications.

Because large amounts of chlorite are detrimental to the use ofdichloric acids according to the invention in the pharmaceutical sector,it is especially advantageous if the end-product of the solutions,according to the invention, do not contain chlorite in more than 20-foldexcess, preferably in not more than 5-fold excess and even morepreferably in not more than a 3-fold excess in percentage by weightrelated to the total weight of the solution.

In particular, the dichloric acids and peroxochlorous acid according tothe invention are present in this solution in volumes of about 0.1-20weight %, preferably 3-5 weight %, related to the percentage by weightof the ClO₂ employed. The qualitative detection is successful usingRaman spectroscopy. The performance of this type of spectroscopy is amatter of course for an expert in this field. The spectrograms which areobtained clearly differ from those which are obtained with the processdescribed in WO 00/48940. The determination of the quantitative sharecan be carried out using titration.

A further qualitative detection is possible using the reaction with theheme iron. In the presence of the dichloric acids of the invention, thetemporal course of the change in intensity of the Soret bands is clearlydifferent to the results of the solutions which were obtained with theprocess described in WO 00/48940.

The process according to the invention consists of a reaction ofchlorine dioxide with an aqueous or water-containing hydrogen peroxide(or another hydroperoxide or peroxide known to an expert, such as e.g.peroxocarbonate, or perborate, or the urea adduct of the hydrogenperoxide) at a pH value of 6.5 or greater, preferably pH 10-12.Preferably, the pH value should be kept at a constant level.

Moreover, surprisingly, it has been shown that peroxochloric acid, whichoccurs as an intermediate product, as well as its anions andderivatives, can also be obtained by the reaction of chlorine dioxidewith other oxidants which contain the peroxo group.

The reaction can be carried out in an aqueous environment or awater-containing environment. For example, as well as water, solventscan be present which are miscible with water such as alcohols oralkanols such as methanol, ethanol or mixtures of these.

Alternatively, other chlorine oxides can be used initially. For example,chlorine monoxide, preferably in its dimeric form (Cl₂O₂), can also beconverted with a hydroperoxide (preferably hydrogen peroxide) to thedesired product. The reaction is successful in the same pH range asstated for chlorine dioxide.

The reaction temperature can be increased for example up to about 50°C.; in purely aqueous systems, the lowest temperature should bepreferably about 0° C. One should not work with chlorine dioxide under+10° C. however, because the chlorine dioxide gas liquefies below thistemperature and deflagration can occur. If additional organic solventsand/or high concentrations of the active reagents are present, thenlower temperatures, i.e. below the freezing temperature of water, can beused. Preferably, work takes place at room temperature.

The chlorine dioxide required for the reaction is available to expertsand can be manufactured in the usual way. For example, it can bemanufactured by the reaction of a chlorite with an acid (e.g. sodiumchlorite with sulphuric acid) or by the reduction of chlorate—forexample with sulphurous acid.

The chlorine dioxide thus obtained can be liberated in the usualmanner—if necessary after removal of traces of chlorine (Granstrom,Marvin L.; and Lee, G. Fred, J. Amer. Water Works Assoc. 50, 1453-1466(1958)).

If the chlorite used to make ClO₂ is contaminated with carbonate, theClO₂ will be contaminated with CO₂ and/or the carbonic acid adductsdescribed in WO 00/48940. In order to absorb the carbon dioxide, the gasstream containing chlorine dioxide and carbon dioxide should be directedthrough a washing bottle filled with a lye. During short contact times,the CO₂ but not the ClO₂ will be absorbed by the lye. It is preferable,however, to remove the carbonate contamination by fractionedcrystallisation of the sodium chlorite which is used. A contamination ofthe peroxochlorate with carbonate can be easily recognized on the Ramanspectrum. Instead of sharp bands at 1051 cm⁻¹, one obtains a double band1069 cm⁻¹ (wide) and the important bands, within the scope of theinvention, at 1051 cm⁻¹ (sharp).

The chlorine dioxide can be transported with an inert gas such asnitrogen or with a rare gas such as argon, however, air or oxygen forthe reaction with the peroxo compound or the hydroperoxide such ashydrogen peroxide or perborate can also be used. For example, it ispossible to make the chlorine dioxide in a first reaction vessel andthen to introduce it with the above mentioned gases or mixtures of theminto a second reaction vessel which contains the peroxo compound(peroxide or hydroperoxide) in an aqueous or water-containing solution.

The pH value of the reaction mixture is kept equal to, or above, 6.5 byadding a base. It is preferable to keep the pH value constant. This canbe carried out either manually or by using a pH “stat”.

The usual organic or inorganic bases can be used such as bases, forexample caustic soda solution or caustic potash solution oralkaline-earth hydroxides as well as ammonia; or organic bases such asnitrogenous bases. Furthermore, the hydroxides from of quaternaryammonium salts in particular alkyl, trialkyl or tetraalkyl ammoniumhydroxide, or zinc hydroxide can also be used.

The content of hydroperoxide in the reaction mixture can, for example,be determined using potentiometric titration with an acid such ashydrochloric acid.

The solutions obtained according to the procedures described above canbe used in both the form in which they were made or in variations ofthis. For example, superfluous hydrogen peroxide can be removed in theusual way, e.g. with a heavy metal compound such as manganese dioxide.Surpluses of the other oxidants can be removed with similar means.

Surpluses of chlorine dioxide (ClO₂) can be removed with H₂O₂. Thisshould take place as soon as possible, otherwise via2ClO₂+2OH⁻->ClO₂ ⁻+ClO₃ ⁻+H₂O

disturbing ClO₃ ⁻with pentavalent chlorine (chlorate) will be formed. Aproduct containing chlorate is however undesirable.

In order to improve the storage life of the reaction product, storage ata high pH value is suitable, for example, pH 10 and above. Theadjustment of this pH value can be carried out with a suitable base—asdescribed previously in the manufacturing procedure.

In the manufacture of solutions which contain the dichloric acids and/orthe salts of these acids, surprisingly, it is possible to expel andcollect the free acid HOOClO, the dichloric acids or the peroxochloricacid out of the mixture containing chlorite ions with an inert gas suchas a noble gas, e.g. argon, or with nitrogen or with the gases oxygen orair, while lowering the pH to below 6, e.g. pH 5, or less. Surprisingly,it has been demonstrated that the yield can be enormously increased ifthe gas stream distance is kept very short and the stream is cooled.

The mixture which forms after the start of step (a) in the manufacturingprocedure described above contains, at first, very high concentrationsof chlorite ions (ClO₂ ⁻). The chlorite content, however, can beconsiderably reduced by the “passing over” in a gas stream in a basicsolution. In this process, all types of chloric acids are expelled asvolatile compounds in protonated (neutral) form. These are however veryinstable. A base is present in the receiving vessel through which thechloric acids are deprotonated and the anions are formed. After thesolution has been adjusted to pH 6-8, and after defined volumes ofchlorite—for example, in the form of sodium chloride—have been added,the anions of the dichloric acids are formed.

Collection can be carried out, for example, in a base, such as analkaline metal base, alkaline-earth metal base or a zinc base ornitrogenous base such as ammonia or an organic amine. It is alsopossible to freeze out the gaseous acids in a cold trap (e.g. at −100 to−190° C.).

Counterions can be all metal cations and organic cations such as thosefrom nitrogenous bases, in particular quaternary ammonium salts. Thechoice of the most suitable cations can be determined from theindividual purpose of use. For pharmaceutical applications, alkalineearth or alkaline metals, preferably Na⁺ or K⁺, or Zn²⁺ are mostsuitable. In technical applications, organic cations, such as cationsfrom nitrogenous bases, in particular alkyl ammonium cations such astrialkyl ammonium cations or especially quaternary ammonium cations canbe used.

It is appropriate and preferable to store the acid and the salts,according to the invention, in the dark and to make aqueous solutionswith high pH values out of them, e.g. with pH values of 10, 11 or 12 andabove, in particular the range of pH 10 to pH 13, in order to ensure along storage life. Depending on the need, the free acid can be regainedfrom such solutions in the manner described previously and, ifnecessary, can be converted to solutions with the desired pH value orinto salts.

The dichloric acids according to the invention, their derivatives oranions and salts of these, can be used as they are, but particularlyalso in aqueous or water-containing solutions, as oxidants for verydifferent medical, cosmetic, technical and agricultural purposes.

Examples for possible test systems are included in the initially namedpublications and patent documents, which are included herein in thisrespect by reference.

An application possibility exists in the use as pharmaceuticalpreparations (medicaments), or for the manufacture of medicaments, whichcan be administered in all possible methods, in particular topically butalso parentally. The medicament can be formulated in the usual way withthe usual pharmaceutically well-tolerated vehicles and diluting agents.

The invention also relates to pharmaceutical preparations whichincorporate the dichloric acids or peroxochlorous acid, respectively,according to the invention, their anions, derivatives or salts as theactive substance and which can be used in particular to treat theillnesses mentioned in the introduction. Especially preferential, arepreparations for enteral administration such as nasal, buccal, rectaland especially oral administration (preferably avoiding the acid of thestomach, e.g. gastric juice-resistant preparations such as capsules orcoated tablets), as well as particularly for local or parenteraltreatment, such as intravenous, intramuscular or subcutaneousadministration to homothermal animals—in particular humans. Thepreparations contain the active substance alone or preferably togetherwith one or more pharmaceutically applicable vehicle materials. Thedosing of the active substance depends on the illness being treated aswell as the species being treated, its age, weight and individualcondition, individual pharmacokinetic circumstances as well as themethod of application. Preferably, the dosage for the enteral orparticularly the parental administration (for example by infusion orinjection) (most favourably in humans) lies in the range of 0.01 to 100pmol/kg, in particular between 0.1 and 100 pmol. Therefore, for example,a person with a bodyweight of 70 kg should receive 1 mg to 1 g/day, inparticular between 8.5 mg and 850 mg/day, administered in one dose orsplit up into several smaller doses. For local application, thepreferable dosage range lies between 0.1 and 10, in particular between0.5 and 5 mL/100 cm² of a 0.1 to 10 millimolar solution (correspondinglymore or less for larger or smaller surfaces—either applied directly orusing, for example, bandages out of impregnated gauze).

Thus the invention also relates to a method—for the prophylactic and/ortherapeutic treatment of the pathological conditions described here, inparticular for the prophylactic and/or therapeutic treatment of diseaseswhere a strengthening of tissue regeneration, an immunomodulation, animprovement of vaccination reaction or a radiation sensitization isindicated and successful, or one or more of these effects, in particularin the treatment of wounds in warm blooded animals—incorporating theadministration of the dichloric acids or peroxochlorous acid,respectively, its anions, derivatives or salts, according to theinvention, in an effective dosage against the aforementioned diseases toa warm blooded animal, e.g. a human being who requires such a treatment.

The invention also relates to a pharmaceutical composition—for theprophylactic, and in particular, for the therapeutic treatment of thedisease conditions described here, preferably for the prophylacticand/or therapeutic treatment of diseases where a strengthening of tissueregeneration, an immunomodulation, an improvement of vaccinationreaction or a radiation sensitization is indicated and successful, orfor one or more of these effects, in particular in the treatment ofwounds, preferably of a warm blooded animal who is suffering from such acondition—which contains dichloric acids or peroxochlorous acid,respectively, its anions, derivatives or salts, according to theinvention, in a prophylactically, or in particular, therapeuticallyeffective dosage against the aforementioned diseases and one or morepharmaceutically applicable vehicle materials.

The invention also relates to a procedure—for the treatment ofpathological conditions preferably for the prophylactic and/ortherapeutic treatment, in particular of a warm blooded animal,especially a human being, where a strengthening of tissue regeneration,an immunomodulation, an improvement of vaccination reaction or aradiation sensitization is indicated and successful, in particular inthe treatment of wounds in warm blooded animals—which incorporates theadministration of the dichloric acids, or peroxochlorous acid,respectively, its anions, derivatives or salts, according to theinvention, in an effective dosage against the aforementioned diseases toa warm blooded animal, e.g. a human being who requires such a treatment.

The invention also relates to the use of the dichloric acids and/or theperoxochlorous acid and their derivatives, anions or salts, according tothe invention, in a procedure for the treatment of an animal or humanbody.

Therefore, the invention also relates to the use of the dichloric acidsand/or the peroxochlorous acid and their derivatives, anions or salts,according to the invention, preferably for prophylactic and/ortherapeutic treatment of diseases, in particular of a warm bloodedanimal, especially a human being, where a strengthening of tissueregeneration, an immunomodulation, an improvement of vaccinationreaction or a radiation sensitization is indicated and successful, inparticular in the treatment of wounds.

The invention also relates to the use or a method for the use of thedichloric acids and/or the peroxochlorous acid and their derivatives,anions or salts, according to the invention, for the (cosmetic) care ofthe skin, for example when a person has a tendency to develop spots andpimples (e.g. acne) or if pimples are present.

Dosage unit forms are e.g. dragees, tablets, ampoules, vials,suppositories or capsules. Further administration forms, in particularfor solutions of the dichloric acids and/or the peroxochlorous acid andtheir derivatives, anions or salts, according to the invention, are e.g.ointments, creams, pastes, gels, foams, mouthwash, drops, sprays andsimilar. The dosage unit forms, e.g. ampoules, tablets or capsules,contain preferably between about 0.05 g to about 1.0 g, in particularfrom 8.5 mg to 850 mg, of a salt of the dichloric acids their anions orderivatives according to the invention with the usual pharmaceuticalvehicle materials.

The pharmaceutical preparations of the invention were essentiallymanufactured in the known manner, e.g. using conventional mixing,granulating, coating, dissolving or lyophilising methods.

In a preferential experimental procedure, a 0.05 to 1 M solution of adichloric acid salt or the peroxochlorous acid and/or a salt of itsderivatives can be dissolved in bidistilled water at a pH equal toor >10, preferably 10 to 13, in particular 12.5. Immediately beforeadministration, this solution is diluted with common salt, sodium orpotassium bicarbonate and bidistilled water to isotonie inconcentrations of about 1-5 mM approaching the physiological pH. Thissolution is suitable for parental, preferably intravenous application.

In order to make a preferential formulation of a drug for topical use,the method of choice is to dissolve the dichloric acids and/or theperoxochlorous acid or their derivatives, according to the invention, assalts in bidistilled water with concentrations in the lower millimolaror in the upper micromolar range—preferably in the concentration rangeof 0.5-5 mM with the pH equal to or >10, in particular 10 to 13, mostpreferably e.g. pH 11.5 and adjust the solution to isotonie withglycerine or common salt or another suitable well-tolerated, preferablyphysiological agent. Before application, a physiological pH is set withHCl. Further additives are possible. In particular, in connection withthe filling of the medicament into plastic containers, such additivesare suitable which can neutralise traces of transition-metals, because,during storage, transition-metals in the walls can be dissolved and cancatalyse a degradation of the active substance. Examples of suchadditives are oligo and polyalcohols, such as ethylene glycol,desferrioxamine or EDTA (e.g. as disodium EDTA). The solution which isobtained in the above manner can also be applied directly to wounds.

The anions of the dichloric acids or peroxochlorous acid according tothe invention are stable, the acids themselves decompose relativelyquickly. Therefore, an active substance stabilisation can be carried outusing the pH. In order to improve tolerance, the active substancesolution can be lowered to an almost physiological pH by buffer dilutionimmediately before use. This is adequate for a deployment of thepharmacological action throughout the body, because this action does notrely on the receptor-ligand interaction of a conventional drug, but itis, as previously stated, related to a fast and irreversible oxidationreaction. The pharmacological action remains in effect as long as thecell and/or its chemically changed structures are present, i.e. it isnot terminated after diffusion of an active substance from a receptor.

Examples for indication fields in which an enhancement of tissueregeneration is successful, either prophylactically or in particulartherapeutically, for the treatment of a pathological condition are theregeneration after physical damage (e.g. traumatic contusions orlacerations, short-wave rays, radioactive radiation) and after chemicaldamage (e.g. through tissue poisons, such as Lost, chemotherapeuticagents). A further application area in this field is the improvement ofwound healing—in particular stubborn so-called “spontaneous”wounds—which occur as a result of a primary disease (e.g. Diabetesmellitus, vascular disorders, immunosuppression or the result of oldage) and which will not heal. Outstanding examples of such disorders arebedsores and chronic varicose ulcers. Here, wound treatment is to beunderstood as treatment of wounds of the skin, mucous membranes andother tissues such as e.g. liver, myocardium or bone marrow.

Because the dichloric acids or peroxochlorous acid according to theinvention are defined compounds, there are no related difficulties innew drug approval.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the cell culture experiments (stimulation ofgrowth of fibroblasts) as shown in Example 3. These were obtained withactive substance concentrations in the culture medium of 100 μM chloriteand 50 μM of reactive chlorine (RC=sum of the anions of all dichloricacids and the peroxochlorous acid).

Here, a growth stimulating effect of 20-25% of the trial solution 1,which includes both dichloric acids according to the invention and alsochlorite, is clearly recognisable in the cell culture which, in relationto the controls, is significantly higher.

The application of the solutions containing only RC or chlorite showsexactly how the controls have no effect whatsoever on the growthbehaviour of the fibroblasts.

FIG. 2 shows the titration of the anions of the peroxo acids (dichloricacid, peroxochlorous acid) present in the solution to determine theconcentration of the acid ions. In FIG. 3, the titration curve derivedfrom FIG. 2 is shown which provides exact concentration determination.

FIGS. 4 and 5 are examples of UV spectra. The UV absorption measurementspermit the determination of the chlorite concentration and show anydissolved free chlorine dioxide which may be present.

FIG. 6 shows a mass spectrum of the product solution whereby theperoxochlorite (mass 83.2) and the anion of the dichloric acid (mass189) were identified.

In FIG. 7, the results of an ion chromatography are shown. The retentiontimes of reference substances are provided in Example 4 part 5. Thedichloric acid is detected at 19.77 min, whereby no chlorate (ClO₃ ⁻) isdetermined which excludes chlorate as the cause of the peak in the massspectrum at 82.3 in FIG. 6.

FIG. 8 shows decay rate kinetics with three bacteria strains, E. coli,S. aureus and P. aeruginosa. In all three strains, a bactericidal actionaccording to DIN 58940 was obtained with the product solution.

DETAILED DESCRIPTION

The following examples provide more details about the invention, theseshould however by no means be understood in a limiting sense.

EXAMPLES Example 1 Preparation of the Dichloric Acids

Carefully, drop for drop, sulphuric acid (96%) is stirred into asolution of 100 g anhydrous sodium chlorite in 200 mL water. Thechlorine dioxide which forms is expelled using a strong gas stream (Ar,N₂ or O₂ or CO₂-free air). The gas stream must be so strong that thecontent of ClO₂ does not exceed 5% (danger of explosion). In order totrap elemental chlorine, the gas stream containing ClO₂ is introducedinto three washing bottles attached to each other which are each filledwith 30 mL of a 2 M NaClO₂ solution at pH 11, in a solution of 15 mL of30% hydrogen peroxide in 35 mL of water, which had previously beenadjusted to pH 12 by adding 4M caustic soda solution. A solution ofsodium perborate or sodium percarbonate or another peroxo compound, suchas e.g. the H₂O₂ adduct of urea can be used instead of hydrogenperoxide. During the introduction of the gas, the pH value is controlledwith a glass electrode. By adding 4M NaOH, the pH value during thereaction can be kept at 12. The hydroperoxide or peroxo compound areexhausted when the inflow of gas leads to a permanent yellow coloring. Adrop of the solution of the oxidant (e.g. H₂O₂) will subsequentlydecolorize the yellow solution again.

While stirring, the solution containing reactive chlorine is drippedinto a solution of 500 g citric acid in 3 liters of water which haspreviously been adjusted to pH 4.5 with 2 M caustic soda solution.During this addition, the reactive chlorine compound which forms isexpelled with a strong gas stream (N₂ or O₂). Preferably, the gas streamshould be cooled. The tube connections should be as short as possible.The gas is collected, for example in three washing bottles which areattached behind each other and which are each filled with 50 mL 0.1 MNaOH.

The contents of the three washing bottles are combined and kept at pH>10.

In order to form the dichloric acids according to the invention, the pHis adjusted to 7—for example with hydrochloric acid—and a 10-fold molarexcess of sodium chlorite is added. For storage, preferably, the pHshould be adjusted to about more than 10 up to about 13.

The total content of reactive chlorine anions is determined bypotentiometric titration with 0.1 M HCl with the usual method known tothe man skilled in the art.

The dichloric acids which are formed are present in solution in amixture with a defined volume of chlorite as well as further reactivechlorine compounds.

The presence of the dichloric acids is detected with Raman spectroscopy.

Example 2 Cultivation of MRC 5 Fibroblasts

Solutions:

Culture Medium for MRC 5:

89 mL IF basal medium

10 mL FCS (foetal calf serum)

1 mL L-glutamine stock solution

IF Basal Medium

The IF basal medium is a 1:1 mixture of IMDM (Iscove's ModifiedDulbecco's Medium) and Ham's F12 medium

L-glutamine Stock Solution

200 mM L-glutamine are dissolved in IF basal medium and sterilized byfiltration.

Cultivation:

The MRC 5 cell line used is seeded in non-gelatine coated cell culturedishes. The subsequent cultivation is carried out in an incubator at 37°C. and 5 vol % CO₂ in a water vapor saturated atmosphere. Every secondto third day, the culture medium is changed and after confluence isreached the cells are passaged with a separation rate of 1:5 to 1:10.

Example 3 Cell Biological Test of Active Substance

Solutions:

Culture Medium for MRC 5:

89 mL IF basal medium

10 mL FCS (Foetal Calf Serum)

1 mL L-glutamine stock solution

Serum-Reduced Culture Medium for MRC-5:

98 mL IF basal medium

1 mL FCS (Foetal Calf Serum)

1 mL L-glutamine stock solution

PBS (Phosphate Buffered Saline):

140 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄ and 1.5 mM KH₂PO₄ are dissolved inwater, whereby a pH value of 7.2-7.4 is set. The solution thus obtainedis sterilized by autoclaving.

Cell Lysis Buffer

0.04% SDS (stock solution 10% SDS)

2×SSC (stock solution 20×SSC)

to obtain 25 mL of finished cell lysis buffer, 5.0 mL 20×SSC and 100 μL10% SDS are filled up to 25 mL with PBS.

DAPI Solution

2 μM DAPI in PBS

Cultivation:

The MRC 5 cells are seeded at 400 cells/cm² in a 24 well cell cultureplate. The subsequent cultivation is carried out in an incubator 37° C.and 5 vol. % CO₂ in a water vapor saturated atmosphere. After 24 hoursof precultivation, the culture medium is suctioned off and the cells arewashed with PBS. The culture medium is then changed to serum-reducedculture medium and the active substances to be tested (the followingtable shows an overview) are added.

After 24, 48 and 72 hours, the proliferation of the cells is determinedby quantification of the cellular DNA in a fluorometer(Novostar-Company: BMG Labtechnologies) after DAPI staining. Here, theincreased fluorescence in the samples is equal to a proliferation of thecells.

The plate to be measured is washed once per well with 500 μl PBS andthen 250 μl PBS is placed in each well. 250 μl lysis buffer are addedand the cells are lysed in a shaker at the lowest setting for 30 min atRT. Subsequently, 500 μl DAPI solution is added and the plate is left tostand for a further 10 min at RT.

The plate is measured at 355 nm ex. and 460 nm em. in the Novostar.Normally, work is carried out with a gain adjustment of 1400-1600.

The multiple determinations are averaged and the error values arecalculated. The data obtained is evaluated graphically.

The following stock solutions are used:

TABLE 2 Combination of the active substances used [Concentration of thestock solutions] Content of chlorite Designation Content of [mM] [mM]Comment Control — — Serum-reduced culture medium Sample 1 40 6 inserum-reduced culture medium Sample 2 40 — in serum-reduced culturemedium Sample 3 — 6 in serum-reduced culture mediumObserved Growth Stimulation of Fibroblasts:

For use in the cell culture, the solutions are first diluted in thegiven culture medium. The results shown in FIG. 1 were obtained withactive substance concentrations of 100 μM chlorite and 50 μM of the RC(=mixture of the dichloric acid and the peroxochlorous acid, accordingto the invention, or the anions thereof).

Here, a growth-stimulating effect of 20-25% of the sample solution 1,which contains both RC and also chlorite, is clearly recognisable andthis is also significantly higher in relation to the control.

Example 4 Analytical Determination of the Solution Obtained from Example1

1) pH measurement:

The pH measurement is made with a single-rod glass electrode. Theproduct content and the position of the equilibrium is dependent on thepH value.

2) Titration with 0.1 M HCl:

The titration serves for example for the quantitative determination ofthe dichloric acid content or also the content of peroxochlorous acid orthe peroxochlorate.

1 mL each of the product solution are titrated potentiometrically with0.1 M hydrochloric acid. The titration curves are recorded (pH vs. mL0.1 M HCl). From the acid consumption measured in the derivation of thetitration curve between pH 8.5 and 4.5, the anion content from thecorresponding acids is determined as a sum.

In a typical result, 1 mL product solution results in a consumption 0.72mL 0.1 M HCl and thus a concentration of 0.072 M.

FIG. 2 shows a recorded titration curve:

The derivation of the titration curve and the determination of theconcentration are shown in FIG. 3.

3) UV-Vis absorption spectrum:

The measurement of the UV spectrum serves the quantification of thechlorite content in the product solution. For comparison, FIGS. 1 and 5show spectra of a chlorite-containing and a chlorite-free productsolution. The chlorite signal is seen at 260 nm; chlorine dioxide, whichoriginates from the process, shows a signal at 360 nm.

The absorption values are determined at 260 nm and 500 nm in 1 cm quartzcuvettes. The ClO₂ ⁻ ion content is determined from the differenceA260-A500 and with the help of the extinction coefficient for chloriteof ε260 nm=140 M⁻¹ cm⁻¹ at 260 nm.

An absorption at 360 nm suggests free chlorine dioxide (ε360 nm=1260M⁻¹cm⁻¹).

4) Mass Spectroscopy

The ESI mass spectrometry was carried out with a Bruker Esquire-LCspectrometer in the standard MS mode. The sample was an aqueous productsolution which had been diluted with methanol before the measurement.The scan range used lay between 30 m/z and 400 m/z with capillary exit−65, voltage and skim −15 V; the spectrum represented an average valuefrom 50 measurements.

The arrow on the right in FIG. 6 points to the signal of the dichloricacid (sum formula: Cl₂O₆ ²⁻), the arrow on the left shows the previouslyunknown peroxochlorite species (sum formula: ClO₃ ⁻).

5) Ion Chromatography

All analyses were carried out with a modular ion chromatography systemfrom the Metrohm company.

Pump: Metrohm IC 709 Pump

Detector: Metrohm 732 IC Detector

Suppressor: Metrohm 753 Suppressor Module

Column: Metrosep A 250

Flow rate: 1 ml/min

Injection volume: 20 μL

Eluent: 1 mM NaOH

Immediately before each measurement, known concentrations of thereference substances were freshly prepared. These were then measuredwith the method described above and with the eluents stated.

Retention times of the reference substances:

Substance Retention time [min] NaCl 13.21 NaClO₂ 12.30 NaClO₃ 16.26NaClO₄ 4.36 NaOH 17.32 Na₂CO₃ 21.98 Na₂Cl₂O₆ 19.77

FIG. 7: In the ion chromatography, the dichloric acid shows a typicalpeak as a retention time of 19.77 min. None of the known referencesubstances could be detected. The ion chromatography confirmed thefindings of the mass spectroscopy. A chlorate-typical peak (NaClO₃,retention time 16.26 min) cannot be detected in the solution preparedaccording to Example 1. Therefore, the peak with the sum formula ClO₃ ⁻in the mass spectroscopy (FIG. 6, mass 83.2) can only be the newperoxochlorous acid or anions thereof.

Example 5 Bactericidal Action of the Solution Obtained from Example 1

Decay kinetics according to DIN 58940

Solution according to Example 1 was used in a 1:10 dilution.

Test organisms: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC 29213

Nutrient medium: Casein peptone—Soya peptone (Ph.Eur.2.6.12)

Bacteria incubation time: 18 h +/−1 h

The result is shown in the following Table 3 as well as in FIG. 8. Thus,the bactericidal action of the solution used has been proved accordingto DIN 58940.

TABLE 3 E. coli NaCl P. aeruginosa NaCl S. aureus NaCl Time log log log(h) cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml 0 1.04E+06 6.02 6.021.58E+05 5.20 5.20 1.98E+06 6.30 6.30 2 5.60E+05 5.75 5.75 8.60E+05 5.935.93 9.00E+05 5.95 5.95 4 5.00E+05 5.70 5.70 6.00E+05 5.78 5.78 1.12E+055.05 5.05 6 8.60E+05 5.93 5.93 5.40E+05 5.73 5.73 9.80E+05 5.99 5.99 24 1.04E+06 6.02 6.02 7.60E+05 5.88 5.88 8.00E+05 5.90 5.90 Trials with 300μg/ml DPOLC P. aeruginosa E. coli DPOLC DPOLC S. aureus DPOLC Time loglog log (h) cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml cfu/ml 0 4.60E+05 5.665.66 1.04E+06 6.02 6.02 1.22E+05 5.09 5.09 2 <20 <1.30 1.30 <20 <1.301.30 2.20E+02 2.34 2.34 4 <20 <1.30 1.30 <20 <1.30 1.30 <20 <1.30 1.30 6<20 <1.30 1.30 <20 <1.30 1.30 <20 <1.30 1.30 24  <20 <1.30 1.30 <20<1.30 1.30 <20 <1.30 1.30

What is claimed is:
 1. An aqueous solution comprising a reactivechlorine compound selected from the group consisting of

salts thereof, and mixtures thereof.
 2. The aqueous solution accordingto claim 1, wherein the total concentration of reactive chlorinecompound is at least 0.01 M.
 3. The aqueous solution according to claim1, wherein the total concentration of reactive chlorine compound isabout 0.05 M to about 1 M.
 4. The aqueous solution according to claim 1,wherein the total concentration of reactive chlorine compound is about 1mM to about 5 mM.
 5. The aqueous solution according to claim 1, whereinthe total concentration of reactive chlorine compound is about 0.5 mM toabout 5 mM.
 6. The aqueous solution according to claim 1, comprising acompound having a peak at about 189 m/z in a mass spectrum.
 7. Theaqueous solution according to claim 6, wherein the peak at about 189 m/zin the mass spectrum has a higher intensity than a peak at about 99 m/zin the mass spectrum.
 8. The aqueous solution according to claim 1,comprising a compound having a peak at about 83.2 m/z in a massspectrum.
 9. The aqueous solution according to claim 8, wherein the peakat about 83.2 m/z in the mass spectrum has a higher intensity than apeak at about 99 m/z in the mass spectrum.
 10. The aqueous solutionaccording to claim 1, comprising a compound having a retention time ofabout 19.77 minutes by ion chromatography, wherein under the same ionchromatography conditions, the reference substance NaCl has a retentiontime of about 13.21 minutes and the reference substance NaClO₃ has aretention time of about 16.26 minutes.
 11. The aqueous solutionaccording to claim 10, wherein a peak having a retention time of about16.26 minutes is not detected by ion chromatography.
 12. The aqueoussolution according to claim 10, wherein a peak having a retention timeof about 15 minutes is detected by ion chromatography.
 13. The aqueoussolution of claim 1, further comprising an additional reactive chlorinecompound selected from the group consisting of peroxochlorous acid offormula O═ClOOH, an anion of peroxochlorous acid of formula O═ClOO⁻,salts thereof, and mixtures thereof.
 14. A pharmaceutical compositionfor treatment of wounds or enhancement of tissue regeneration comprisingan effective dosage of an aqueous solution according to claim
 1. 15. Thepharmaceutical composition according to claim 14 formulated forparenteral or topical administration.